Abstract
BackgroundDifferentially methylated regions (DMRs) are associated with many imprinted genes. In mice methylation at a DMR upstream of the H19 gene known as the Imprint Control region (IC1) is acquired in the male germline and influences the methylation status of DMRs 100 kb away in the adjacent Insulin-like growth factor 2 (Igf2) gene through long-range interactions. In humans, germline-derived or post-zygotically acquired imprinting defects at IC1 are associated with aberrant activation or repression of IGF2, resulting in the congenital growth disorders Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, respectively. In Wilms tumour and colorectal cancer, biallelic expression of IGF2 has been observed in association with loss of methylation at a DMR in IGF2. This DMR, known as DMR0, has been shown to be methylated on the silent maternal IGF2 allele presumably with a role in repression. The effect of IGF2 DMR0 methylation changes in the aetiology of BWS or SRS is unknown.Methodology/Principal FindingsWe analysed the methylation status of the DMR0 in BWS, SRS and Wilms tumour patients by conventional bisulphite sequencing and pyrosequencing. We show here that, contrary to previous reports, the IGF2 DMR0 is actually methylated on the active paternal allele in peripheral blood and kidney. This is similar to the IC1 methylation status and is inconsistent with the proposed silencing function of the maternal IGF2 allele. Beckwith-Wiedemann and Silver-Russell patients with IC1 methylation defects have similar methylation defects at the IGF2 DMR0, consistent with IC1 regulating methylation at IGF2 in cis. In Wilms tumour, however, methylation profiles of IC1 and IGF2 DMR0 are indicative of methylation changes occurring on both parental alleles rather than in cis.Conclusions/SignificanceThese results support a model in which DMR0 and IC1 have opposite susceptibilities to global hyper and hypomethylation during tumorigenesis independent of the parent of origin imprint. In contrast, during embryogenesis DMR0 is methylated or demethylated according to the germline methylation imprint at the IC1, indicating different mechanisms of imprinting loss in neoplastic and non-neoplastic cells.
Highlights
Aberrant imprinting of the Insulin-like growth factor 2 (IGF2) gene plays a role in the pathogenesis of the overgrowth disorder Beckwith-Wiedemann syndrome (BWS, OMIM#130650), the growth-restriction condition Silver-Russell syndrome (SRS, OMIM#180860), as well as various human cancers including Wilms tumour, rhabdomyosarcoma, hepatoblastoma, colorectal and breast carcinomas [1,2,3,4,5,6]
IGF2 DMR0 methylation status in BWS and SRS patients We examined the methylation status of DMR0 in peripheral blood leukocyte DNA derived from BWS patients with IC1 hypermethylation (n = 7), IC2 hypomethylation (n = 37) or normal methylation at both IC1 and IC2 (n = 27) and SRS patients with IC1 hypomethylation (n = 2), using bisulphite and pyrosequencing analysis
Patients with normal methylation at IC1 or hypomethylation at IC2 had normal methylation levels at DMR0 (Median methylation 53%; IQR 48.2%, 57.1%). These results suggest that either methylation changes in BWS and SRS were occurring in trans or that methylation at the DMR0 is on the paternal allele
Summary
Aberrant imprinting of the Insulin-like growth factor 2 (IGF2) gene plays a role in the pathogenesis of the overgrowth disorder Beckwith-Wiedemann syndrome (BWS, OMIM#130650), the growth-restriction condition Silver-Russell syndrome (SRS, OMIM#180860), as well as various human cancers including Wilms tumour, rhabdomyosarcoma, hepatoblastoma, colorectal and breast carcinomas [1,2,3,4,5,6]. Mouse models have been used to demonstrate that the imprinted expression of the closely linked Igf and H19 genes is controlled by specific differentially methylated regions (DMRs) [7,8,9,10]. The molecular basis of BWS is heterogeneous with 5% of patients exhibiting gain of methylation at IC1 with biallelic activation of IGF2 and biallelic silencing of H19 Another group of BWS patients has normal IC1 methylation but abnormal methylation at IC2, a maternally methylated imprinting control region within the KCNQ1 and KCNQ1OT1 gene cluster which seems to operate independent of IGF2 and H19 [2]. These results imply IGF2 imprinting defects in congenital growth disorders and Wilms tumours arise through different epigenetic mechanisms
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